JP2022079062A - Method for inserting exogenous gene onto chromosome of animal cell, animal cell, kit for inserting exogenous gene, vector, guide rna, and guide rna expression vector - Google Patents

Abstract

To provide a method for inserting an exogenous gene onto a chromosome of an animal cell, which can achieve uniform expression of the exogenous gene; an animal cell; and a kit for inserting exogenous gene.SOLUTION: A method for inserting an exogenous gene onto a chromosome of an animal cell includes the step of inserting an exogenous gene to the upstream region of a Zxdb gene of an X chromosome of an animal cell. The animal cell has a recombinase recognition sequence inserted to the upstream region of the Zxdb gene of the X chromosome. A kit for inserting exogenous gene has: the animal cell; a recombinase that recognizes the recombinase recognition sequence inserted to the chromosome of the animal cell; and a vector having the same recombinase recognition sequence as the recombinase recognition sequence inserted to the chromosome of the animal cell.SELECTED DRAWING: None

Description

The present invention relates to a method for inserting a foreign gene onto the chromosome of an animal cell, an animal cell, a kit for inserting a foreign gene, a vector, a guide RNA, and a guide RNA expression vector.

Conventionally, in order to analyze the function of an unexplained gene in a cell, the cDNA of the gene or the shRNA for the gene has been integrated into a chromosome and expressed. If these nucleic acids are randomly integrated into the chromosome, they may not be expressed or may be weakly expressed depending on the integration position, and there is a risk of destroying existing genes.

On the other hand, a chromosome locus called a safe harbor locus has been searched so far. The safe harbor locus is a site where a uniform and constant level of gene expression occurs at a position that is not harmful even if a foreign gene is inserted. Safe harbor loci such as Rosa26 found in mouse cells are commonly used as target locations for inserting foreign genes.

Beard C et al., “Efficient Method to Generate Single-Copy Transgenic Mice by Site-Specific Integration in Embryonic Stem Cells.”, Genesis, Vol. 44, pp. 23-28, 2006.

However, in recent years, it has been pointed out that the expression of the foreign gene placed in the relevant position does not occur uniformly (see, for example, Reference 1). In particular, a foreign gene to be expressed by a tetracycline-dependent promoter undergoes a phenomenon called silencing, and the expression level becomes a mosaic that varies depending on individual cells, and uniform expression cannot be achieved.

The present invention has been made in view of the above circumstances, and provides a method for inserting a foreign gene onto a chromosome of an animal cell, which can achieve uniform expression of the foreign gene. Also provided are animal cells capable of achieving uniform expression of foreign genes, foreign gene insertion kits, vectors, guide RNAs, and guide RNA expression vectors.

As a result of diligent research to achieve the above object, the present inventors have inserted a foreign gene at a position about 10.6 kbp upstream from the transcription start point of the Zxdb gene on the X chromosome of an animal cell, thereby causing the foreign gene. We have found that the gene is uniformly expressed, and have completed the present invention.

That is, the present invention includes the following aspects.
(1) A method for inserting a foreign gene onto an animal cell chromosome, which comprises a step of inserting the foreign gene into the upstream region of the Zxdb gene of the X chromosome of the animal cell.
(2) The insertion method according to (1), wherein the upstream region of the Zxdb gene is a region 1 to 50 kbp upstream from the transcription start site of the Zxdb gene.
(3) The insertion method according to (1) or (2), wherein the upstream region of the Zxdb gene is a region 5 to 15 kbp upstream from the transcription start site of the Zxdb gene.
(4) The insertion method according to any one of (1) to (3), wherein the upstream region of the Zxdb gene is a region 9 to 11 kbp upstream from the transcription start site of the Zxdb gene.
(5) An animal cell in which a recombinase recognition sequence is inserted in the upstream region of the Zxdb gene on the X chromosome.
(6) The animal cell according to (5), wherein the recombinase recognition sequence is an FRT sequence or a loxP sequence.
(7) The animal cell according to (5) or (6), which is a cell derived from human or mouse.
(8) The animal cell according to any one of (5) to (7) and
A recombinase that recognizes the recombinase recognition sequence inserted into the chromosome of the animal cell,
A vector having the same recombinase recognition sequence as the recombinase recognition sequence inserted into the chromosome of the animal cell,
A kit for inserting foreign genes.
(9) Foreign genes and
A nucleic acid having a sequence homologous to the upstream region of the Zxdb gene on the X chromosome of the target animal cell bound upstream and downstream of the foreign gene.
Including the vector.
(10) A nucleic acid having a sequence homologous to the sequence represented by SEQ ID NO: 1 is bound upstream of the foreign gene, and homologous to the sequence represented by SEQ ID NO: 2 downstream of the foreign gene. The vector according to (9), wherein a nucleic acid having a different sequence is bound.
(11) A guide RNA containing a nucleic acid consisting of the base sequence represented by SEQ ID NO: 3.
(12) A guide RNA expression vector containing a nucleic acid consisting of the base sequence represented by SEQ ID NO: 4.

According to the insertion method, animal cells, foreign gene insertion kit, vector, guide RNA, and guide RNA expression vector of the above-described embodiment, uniform expression of the foreign gene can be achieved.

It is a block diagram of the plasmid for the acquisition of zeocin resistance in Example 1. It is a block diagram of the GFP expression plasmid in Example 1. FIG. It is a figure which shows the insertion position of the expression unit on the X chromosome identified by the genome walking method in Example 1. FIG. It is a bright field image (left) and a fluorescence image (right) of MIN6 cells having different insertion positions on the chromosome of the GFP expression unit in Example 2. It is a block diagram of the DsRed2 expression plasmid in Example 3. FIG. It is a block diagram of the glucokinase expression plasmid in Example 4. 3 is a graph showing the amount of insulin secreted in MIN6 cells having different insertion positions on the chromosome of the glucokinase expression unit in Example 4. It is a block diagram of the donor plasmid (zeocin expression plasmid) and the guide RNA expression plasmid in Example 5. It is a figure which shows the target sequence of the primer for confirming the insertion position on the chromosome of the DNA fragment (including the cDNA of zeocin) derived from the donor plasmid in Example 5. It is a block diagram of the GFP expression plasmid in Example 5. The bright field image (left) and the fluorescence image (right) of the MIN6 cell in which the GFP expression unit in Example 5 was inserted into the upstream region of the Zxdb gene on the X chromosome.

≪Definition of terms≫
As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a nucleotide polymer in which nucleotides are bound by phosphodiester bonds. The "polynucleotide" and "nucleic acid" may be DNA, RNA, or may be composed of a combination of DNA and RNA. Further, the "polynucleotide" and "nucleic acid" may be a polymer of a natural nucleotide, and the natural nucleotide and the unnatural nucleotide (an analog of the natural nucleotide, a base portion, a sugar portion and a phosphate portion at least one portion thereof) may be used. It may be a polymer with a nucleotide modified with (eg, a phosphorothioate skeleton), or it may be a polymer of an unnatural nucleotide.
In the present specification, the base sequence of "polynucleotide" or "nucleic acid" is described by a generally accepted one-letter code unless otherwise specified. Unless otherwise specified, the base sequence is described from the 5'side to the 3'side.
In the present specification, the nucleotide residues constituting the "polynucleotide" or "nucleic acid" may be simply described by adenine, thymine, cytosine, guanine, uracil, etc., or a one-letter code thereof.

As used herein, the term "gene" refers to a polynucleotide comprising at least one open reading frame encoding a particular protein. The gene can include both exons and introns.

As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to a polymer of amino acids linked by an amide bond. The "polypeptide", "peptide" or "protein" may be a polymer of natural amino acids, or may be a polymer of natural amino acids and unnatural amino acids (chemical analogs of natural amino acids, modified derivatives, etc.). It may be a polymer of unnatural amino acids. Unless otherwise specified, the amino acid sequence is described from the N-terminal side to the C-terminal side.

As used herein, the term "genome editing" refers to inducing a mutation at a desired location (target region) on the genome. Genome editing may include the use of nucleases engineered to cleave the DNA of the target region. Typically, the use of site-specific nucleases induces double-strand breaks (DSBs) in the DNA of the target region, followed by homologous directed repair (HDR) and non-homologous end recombination (Non-). The genome is repaired by an endogenous process of the cell, such as Homology End-Joining Repair (NHEJ). NHEJ is a repair method in which double-stranded cut ends are ligated without using a repair template DNA, and insertion and / or deletion (indel) is frequently induced during repair. HDR is a repair mechanism using a repair template DNA, and it is also possible to introduce a desired mutation into a target region.

As used herein, the term "donor vector" refers to exogenous DNA used as a template DNA for repair. The donor vector contains a base sequence adjacent to the target region as a homology arm. In the present specification, the homology arm consisting of the base sequence adjacent to the 5'side of the target region is referred to as "5'arm", and the homology arm consisting of the base sequence adjacent to the 3'side of the target sequence is referred to as "3'arm". May be stated. The donor vector can contain the desired base sequence between the 5'arm and the 3'arm. The length of each homology arm is usually about 0.1 kbp to 10 kbp. The lengths of the 5'arm and the 3'arm may be the same or different, but are preferably the same.

As used herein, the term "Cas protein" refers to a CRISPR-associated protein. In a preferred embodiment, the Cas protein forms a complex with a guide RNA and exhibits endonuclease activity or nickase activity. The Cas protein is not particularly limited, and examples thereof include Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, and C2c3 protein. Cas proteins include wild-type Cas proteins and their homologs (paralogs and orthologs), as well as variants thereof, as long as they exhibit endonuclease activity or nickase activity in conjunction with guide RNA.
In a preferred embodiment, the Cas protein is involved in a class 2 CRISPR / Cas system, more preferably a type II CRISPR / Cas system. A preferred example of the Cas protein is the Cas9 protein.

As used herein, the term "Cas9 protein" refers to a Cas protein involved in the type II CRISPR / Cas system. The Cas9 protein forms a complex with the guide RNA and exhibits the activity of collaborating with the guide RNA to cleave the DNA of the target region. Cas9 protein includes wild-type Cas9 protein and its homologs (paralogs and orthologs) and variants thereof, as long as they have the above-mentioned activity. The wild-type Cas9 protein has a RuvC domain and an HNH domain as nuclease domains, but the Cas9 protein herein may be one in which either the RuvC domain or the HNH domain is inactivated.
The organism species from which the Cas9 protein is derived is not particularly limited, but bacteria belonging to the genus Streptococcus, Staphylococcus, Neisseria, Treponema and the like are preferably exemplified. More specifically, S. pyogenes, S. streptococcus. thermophilus, S.A. aureus, N. et al. Meningitidis, or T.I. Cas9 protein derived from dentalcola and the like is preferably exemplified. In a preferred embodiment, the Cas9 protein is S. It is a Cas9 protein derived from pyogenes.

Information on the amino acid sequences of various Cas proteins and their coding sequences can be obtained on various databases such as GenBank and UniProt. For example, S. As the amino acid sequence of Cas9 protein of pyogenes, those registered in UniProt as accession number Q99ZW2 can be used.

As used herein, the terms "guide RNA" and "gRNA" are used interchangeably to refer to RNA that can form a complex with a Cas protein and induce the Cas protein to a target region. In a preferred embodiment, the guide RNA comprises a CRISPR RNA (crRNA) and a transactivated CRISPR RNA (tracrRNA). The crRNA is involved in binding to a target region on the genome, and the tracrRNA is involved in binding to the Cas protein. In a preferred embodiment, the crRNA comprises a spacer sequence and a repeat sequence, the spacer sequence binding to the complementary strand of the target sequence in the target region. In a preferred embodiment, the tracrRNA comprises an anti-repeat sequence and a 3'tail sequence. The anti-repeat sequence has a sequence complementary to the repeat sequence of crRNA and forms a base pair with the repeat sequence, and the 3'tail sequence usually forms three stem loops.
The guide RNA may be a single guide RNA (sgRNA) in which the 3'end of the crRNA is linked to the 5'end of the tracrRNA, and the crRNA and the tracrRNA are separate RNA molecules, and base pairs are used in repeat sequences and anti-repeat sequences. May be formed.

As used herein, the term "target sequence" refers to a DNA sequence in the genome that is the subject of cleavage by the Cas protein. When using the Cas9 protein as the Cas protein, the target sequence needs to be a sequence adjacent to the 5'side of the protospacer adjacent motif (PAM). As the target sequence, a sequence of 17 to 30 bases (preferably 18 to 25 bases, more preferably 19 to 22 bases, still more preferably 20 bases) adjacent immediately before the 5'side of PAM is selected. A known design tool such as CRISPR DESIGN (crispr.mit.edu/) can be used to design the target sequence.

As used herein, the terms "protospacer flanking motif" and "PAM" are used interchangeably and refer to sequences recognized by the Cas protein upon DNA cleavage by the Cas protein. The sequence and position of PAM depends on the type of Cas protein. For example, in the case of Cas9 protein, the PAM needs to be adjacent immediately after the 3'side of the target sequence. The sequence of PAM corresponding to the Cas9 protein depends on the bacterial species from which the Cas9 protein is derived. For example, S. The PAM corresponding to the Cas9 protein of pyogenes is "NGG" and S. streptococcus. The PAM corresponding to the Cas9 protein of thermophilus is "NNAGAA", and S.I. The PAM corresponding to the Cas9 protein of aureus is "NNGRRT" or "NNGRR (N)". The PAM corresponding to the Cas9 protein of meningitidis is "NNNNGATT", which is T.I. It is "NAAAAC" corresponding to the Cas9 protein of detentola ("R" is A or G; "N" is A, T, G or C).

As used herein, the term "expressible state" refers to a state in which the polynucleotide can be transcribed in the cell into which the polynucleotide has been introduced.
As used herein, the term "expression vector" refers to a vector containing a target polynucleotide, which comprises a system for making the target polynucleotide expressible in a cell into which the vector has been introduced. For example, the “Cas protein expression vector” means a vector capable of expressing the Cas protein in the cell into which the vector has been introduced. Further, for example, the “guide RNA expression vector” means a vector capable of expressing the guide RNA in the cell into which the vector has been introduced.

<< How to insert a foreign gene onto the chromosome of an animal cell >>
The method for inserting a foreign gene onto the chromosome of an animal cell according to an embodiment of the present invention (hereinafter, may be simply referred to as “method for inserting the present embodiment”) is upstream of the Zxdb gene on the X chromosome of the animal cell. Includes the step of inserting a foreign gene into the region.

In an experimental system using MIN6 cells, which are insulin-secreting cell lines derived from mice, the inventors set the insertion position on the chromosome on which the foreign gene can be uniformly expressed from the transcription start site of the Zxdb gene on the X chromosome. The position upstream of about 10.6 kbp was identified, and the present invention was completed. That is, the insertion position of the upstream region of the Zxdb gene on the X chromosome identified by the inventors can be said to be a novel safe harbor locus.
According to the insertion method of this embodiment, as shown in Examples described later, the foreign gene integrated on the chromosome can be uniformly expressed under in vitro conditions.
The term "uniformly expresses a foreign gene" as used herein means that in a cell in which the foreign gene is integrated on a chromosome, the expression level of the foreign gene does not vary between cells and the foreign gene is expressed. Means.

Hereinafter, each step of the insertion method of the present embodiment will be described in detail.

<Insert process>
In the insertion step, a foreign gene is inserted into the upstream region of the Zxdb gene on the X chromosome of an animal cell.

The upstream region of the Zxdb gene on the X chromosome means a region between the Zxdb gene and the Gspt2 gene present upstream thereof, specifically, a region 1 bp to 86 kbp upstream from the transcription start site of the Zxdb gene. Among them, the insertion position of the foreign gene is preferably a region 1 to 50 kbp upstream from the transcription start site of the Zxdb gene, and more preferably a region 5 to 15 kbp upstream from the transcription start point of the Zxdb gene. It is more preferably a region upstream of ~ 11 kbp, even more preferably a region upstream of 10 ~ 10.8 kbp, particularly preferably a region upstream of 10.5-10.7 kbp, 10.6 kbp upstream. Most preferably it is a region.

The base sequence information of the Zxdb gene can be obtained on various databases such as GenBank and UniProt. For example, as the nucleotide sequence information of the mouse Zxdb gene, those registered in GenBank as accession number NC_0000866.8 can be used. As the nucleotide sequence information of the mRNA of the mouse Zxdb gene, the one registered in GenBank as NM_001081473.1 can be used. By comparing the base sequence information of the mouse Zxdb gene with the base sequence information of the mRNA of the mouse Zxdb gene, the transcription start point can be confirmed, and the upstream region of the transcription start point can be used as the insertion position of the foreign gene. can.
Further, as the base sequence information of the human Zxdb gene, those registered in GenBank as accession number NC_00000231.11 can be used. As the base sequence information of the mRNA of the human Zxdb gene, the one registered in GenBank as NM_007157.4 can be used. By comparing the base sequence information of the human Zxdb gene with the base sequence information of the mRNA of the human Zxdb gene, the transcription start point can be confirmed, and the upstream region of the transcription start point can be used as the insertion position of the foreign gene. can.

In addition, the sequence identity (or homology) between animal species of the base sequence of the upstream region of the Zxdb gene of the X chromosome is such that the two base sequences are inserted and deleted so that the corresponding bases match most. It is determined as the ratio of matching bases to the entire base sequence, excluding the gaps in the obtained alignment, by juxtaposing them with gaps in the portions corresponding to. The sequence identity between the base sequences can be determined by using various homology search software known in the art. For example, the sequence identity value of the base sequence can be obtained by calculation based on the alignment obtained by the known homology search software BLASTN. In addition, using the above method, it is possible to identify a position in another animal species corresponding to a position about 10.6 kbp upstream from the transcription start point of the Zxdb gene on the X chromosome in the mouse.

[Animal cells]
The animal species from which the animal cells are derived may be any animal species having the Zxdb gene on the X chromosome, and among them, mammals are preferable. Examples of mammals include humans, mice, rats, dogs, cats, rabbits, pigs, cows, horses, monkeys, marmosets, chickens, sheep, goats and the like. Among them, as a mammal, human or mouse is preferable. That is, the animal cells used in the insertion method of the present embodiment are preferably cells derived from humans or mice.

The type of cell is not particularly limited, but cells derived from various tissues or having various properties, specifically, for example, blood cells, hematopoietic stem cells / precursor cells, spouses (eggs), fertilized eggs, fibroblasts, epithelium. Examples thereof include cells, vascular endothelial cells, nerve cells, hepatocytes, keratin-producing cells, muscle cells, epidermal cells, endocrine cells, tissue stem cells, iPS cells, ES cells, cancer cells and the like. Further, for example, cells having various diseases such as β cells derived from type 1 or type 2 diabetic patients can be mentioned.

[Foreign gene]
The foreign gene is not particularly limited, and a desired gene that is to be integrated and expressed on the chromosome of a cell can be used.
For example, as shown in Examples described later, by inserting a glucokinase gene (glucokinase cDNA) into MIN6 cells, which are insulin-secreting cell lines derived from mice, cells with enhanced insulin-secreting ability can be obtained. can.

The size of the foreign gene is preferably 0.1 kbp or more and 10 kbp or less, more preferably 0.1 kbp or more and 10 kbp or less, and 0.1 kbp or more and 8 kbp or less from the viewpoint of insertion efficiency and ease of confirmation of the inserted foreign gene. More preferably, 0.1 kbp or more and 5 kbp or less are particularly preferable.

The foreign gene consists of a functional gene sequence, but one or more selected from the group consisting of a promoter sequence, a transcription termination sequence, a marker gene, a tag sequence, and a recombinase recognition sequence may be further added. ..

The promoter sequence is not particularly limited, and for example, various pol II promoters can be used. The pol II promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, a β-actin promoter, a CAG promoter, and a CBh promoter.

The "marker gene" refers to a gene that enables selection and selection of cells by introducing the marker gene into cells. Specific examples of the marker gene include a drug resistance gene, a fluorescent protein gene, a luminescent enzyme gene, a color-developing enzyme gene, and the like. These may be used alone or in combination of two or more. Examples of the drug resistance gene include puromycin resistance gene, neomycin resistance gene, tetracycline resistance gene, canamycin resistance gene, zeosin resistance gene, hyglomycin resistance gene, chloramphenicol resistance gene, histidenol resistance gene, and blastsaidin. Examples include resistance genes. Examples of the fluorescent protein gene include a green fluorescent protein (GFP) gene, a yellow fluorescent protein (YFP) gene, a red fluorescent protein (RFP) gene and the like. Examples of the luminescent enzyme gene include a luciferase gene and the like. Examples of the color-developing enzyme gene include β-galactosidase gene, β-glucuronidase gene, alkaline phosphatase gene and the like.

Examples of the tag sequence include affinity tags, degron tags, localized tags, and the like.

The foreign gene may be used in the form of a vector.
Examples of the vector include a plasmid vector and a virus vector.

Examples of the virus vector include a retrovirus (including lentivirus) vector, an adenovirus vector, an adeno-associated virus vector, a Sendai virus vector, a herpes virus vector, a vaccinia virus vector, a pox virus vector, a poliovirus vector, and a silvis virus vector. , Rabdovirus vector, paramixovirus vector, orthomixovirus vector and the like.

Examples of the plasmid vector include, but are not limited to, pBluescript, pDrive, pX459, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo and the like.

Above all, a plasmid vector is preferable.

Further, the foreign gene is preferably in the form of a donor vector to which a nucleic acid (homology arm) of an arbitrary length having a sequence homologous to the upstream region of the Zxdb gene on the X chromosome is bound upstream and downstream thereof. That is, it is preferable that the vector contains a foreign gene and a nucleic acid (homology arm) having a sequence homologous to the upstream region of the Zxdb gene of the X chromosome of the target animal cell bound upstream and downstream of the foreign gene. ..

The 5'arm that binds upstream of the foreign gene is preferably a nucleic acid having a sequence homologous to the sequence represented by SEQ ID NO: 1.
The 3'arm that binds upstream of the foreign gene is preferably a nucleic acid having a sequence homologous to the sequence represented by SEQ ID NO: 2.

The donor vector may be a circular DNA vector (for example, a plasmid vector) or a linear DNA vector.

[Insert method]
Foreign genes can be inserted using known genome editing techniques.

As the known genome editing technology, any genome editing technology that induces HDR may be used, and for example, a CRISPR-Cas9 system, a TALEN system, and a Zn finger nuclease system can be used. Of these, it is preferable to use the CRISPR-Cas9 system.

(TALEN system)
The TALEN system is a system using a Transcription activator-like effector nuclease (TALEN), which is a fusion of a customized binding domain and a non-specific FokI nuclease domain. The DNA-binding domain consists of conserved repeats derived from transcription activator-like effectors (TALE), a protein secreted by Pseudomonadota proteobacterium that alters gene transcription in host plants. To insert a foreign gene into a target site of the genome by the TALEN system, TALEN is prepared for the insertion site in the genome, and a donor vector having the foreign gene and having homology around the insertion site is prepared. By co-introducing TALEN and the donor vector, the target site is cleaved, and subsequently, the target foreign DNA is introduced by HDR using the donor vector. By having the foreign gene have a drug selection marker, it is possible to efficiently select cells in which the foreign gene has been introduced by drug selection.

(CRISPR-Cas9 system)
The CRISPR-Cas9 system is a system that utilizes a locus that functions as a kind of acquired immunity against nucleic acids (viral DNA, viral RNA, plasmid DNA) that have invaded from the outside, which are possessed by bacteria and paleobacteria. In order to insert a foreign gene into a target site of the genome by the CRISPR-Cas9 system, a guide RNA or an expression vector thereof for inducing Cas9 at the target site and Cas9 or an expression vector thereof are required.

The guide RNA can be designed by appropriately setting a target sequence from the sequence in the upstream region of the Zxdb gene on the X chromosome and using a method known as a nucleic acid containing a sequence complementary to the target sequence. Among them, the guide RNA preferably contains a nucleic acid consisting of the base sequence represented by SEQ ID NO: 3. The nucleotide sequence represented by SEQ ID NO: 3 is a sequence complementary to the target sequence, with the region upstream of 10,604 to 10,623 bp from the transcription start site of the Zxdb gene as the target sequence.

When the guide RNA is in the form of an expression vector, the expression vector preferably contains a sequence encoding the guide RNA and a promoter that controls the expression of the sequence encoding the guide RNA. In the expression vector, the guide RNA coding sequence is functionally linked to the promoter.

The promoter is not particularly limited, and for example, a pol II promoter can be used, but a pol III promoter is preferable from the viewpoint of more accurately transcribing a relatively short RNA. The pol III promoter is not particularly limited, and examples thereof include a mouse and human U6-snRNA promoter, a human H1-RNase P RNA promoter, and a human valine-tRNA promoter. When using the U6 promoter, it is preferred that the 5'end of the guide RNA is "G" for transcription initiation.

In addition to the coding sequence and promoter of the guide RNA, the expression vector contains an enhancer, a poly A addition signal, a marker gene, an origin of replication, a gene encoding a protein that binds to the origin of replication and controls replication, and the like. You may be.

The type of expression vector is not particularly limited, and a known expression vector can be used. Examples of the expression vector include a plasmid vector and a virus vector. Examples of the plasmid vector and the viral vector include the same as those exemplified in the above-mentioned "foreign gene".

Preferred expression vectors include guide RNA expression vectors containing nucleic acids consisting of the nucleotide sequence represented by SEQ ID NO: 4. The nucleotide sequence represented by SEQ ID NO: 4 is a sequence of a region upstream of 10,604 to 10,623 bp from the transcription start site of the Zxdb gene.

The Cas protein is not particularly limited as long as it is used in the CRISPR / Cas system. For example, various substances that can form a complex with a guide RNA and are guided to the target region by the guide RNA to cleave the DNA of the target region by double strands can be used.

An expression vector of Cas protein may be used instead of Cas protein.
The Cas protein expression vector preferably contains a Cas protein coding sequence and a promoter that controls the expression of the Cas protein coding sequence. In the expression vector, the Cas protein coding sequence is functionally linked to the promoter.
Examples of the promoter include those similar to those exemplified in the above-mentioned "foreign gene".

In addition to the Cas protein coding sequence and promoter, the expression vector contains an enhancer, a poly A addition signal, a marker gene, an origin of replication, a gene encoding a protein that binds to the origin of replication and controls replication, and the like. You may be.

The type of expression vector is not particularly limited, and a known expression vector can be used. Examples of the expression vector include a plasmid vector and a virus vector. Examples of the plasmid vector and the viral vector include the same as those exemplified in the above-mentioned "foreign gene".

The Cas protein coding sequence contained in the expression vector may be codon-optimized depending on the species from which the cell into which the expression vector is introduced is derived. In general, codon optimization refers to replacing at least one codon in the original base sequence with a codon that is more frequently used in the species of interest, while preserving the original amino acid sequence. The codon usage frequency table is easily available, for example, in "Codon Usage Database" (www.kazusa.or.jp/codon/) provided by the Kazusa DNA Research Institute, and using these tables, the codon usage frequency table can be easily obtained. Codons can be optimized. Computer algorithms for codon-optimizing specific sequences for expression in specific animal species are also available, for example, in Gene Forge (Aptagen; Jacobus, PA) and the like.

The expression vector of the guide RNA and the expression vector of the Cas protein may be the same expression vector. That is, in the insertion method of the present embodiment, an expression vector containing a guide RNA coding sequence and a Cas protein coding sequence in an expressible state can be used. In the expression vector, it is preferable that the guide RNA coding sequence and the Cas protein coding sequence are functionally linked to different promoters.

By co-introducing a donor vector having a foreign gene and having homology around the insertion site with the guide RNA or its expression vector and Cas9 or its expression vector, the target site is cleaved and subsequently the donor. The target foreign gene is introduced by HDR using a vector. By having the foreign gene have a drug selection marker, it is possible to efficiently select cells in which the foreign gene has been introduced by drug selection.

The donor vector having the foreign gene, the guide RNA or its expression vector, and the Cas9 or its expression vector can be used as a foreign gene insertion kit. That is, in one embodiment, the present invention provides a foreign gene insertion kit comprising a donor vector having the foreign gene, a guide RNA or an expression vector thereof, and Cas9 or an expression vector thereof.

(Zinc finger nuclease (ZFN) system)
The Zn finger nuclease (ZFN) system is a genome editing using an artificial restriction enzyme consisting of a Zn finger domain and a DNA cleavage domain. One Zn finger domain recognizes 3 bases, but by linking 3 to 5 Zn finger domains, a DNA-binding protein that recognizes a specific 9 to 15 base sequence can be designed. By fusing this DNA-binding protein with the FokI nuclease domain, an artificial nuclease ZFN that cleaves the target site is designed. To insert the plasmid into the target site of the genome by the ZFN system, a ZFN is prepared for the insertion site in the genome, and a donor vector having homology around the insertion site is prepared. By co-introducing these vectors, the target site is cleaved, and subsequently, the target foreign DNA is introduced by HDR using the donor vector. By having the foreign DNA have a drug selection marker, it is possible to efficiently select cells in which foreign DNA has been introduced by drug selection.

As a method for introducing a foreign gene or the above-mentioned tool of the known genome editing technique into a cell, it is possible to appropriately select it according to the target cell and the type of material (whether it is a nucleic acid or a protein, etc.). can.

Examples of the method for introducing the vector into cells include a lipofection method, a microinjection method, a DEAE dextran method, a gene gun method, an electroporation method, a calcium phosphate method and the like. When the vector is a viral vector, examples of the method for infecting cells with the viral vector include the polybrene method.

The method for introducing RNA into cells is not particularly limited, and known methods can be appropriately selected and used. For example, as RNA, a commercially available RNA transfection reagent such as Lipofectamine (registered trademark) MessengerMAX (manufactured by Life Technologies) can be used.

The method for introducing the protein into the cell is not particularly limited, and a known method can be appropriately selected and used. Examples of such a method include a method using a protein-introducing reagent, a method using a protein-introduced domain (PTD) fusion protein, a microinjection method, and the like.

The foreign gene and the above-mentioned tools of the known genome editing technique may be introduced into cells at the same time, sequentially, or separately after a certain period of time. Above all, it is preferable to introduce all of these into cells at the same time.

<Arbitrary process>
[Culture process]
The insertion method of the present embodiment may further include a culture step of culturing the cells after the insertion step, specifically, after introducing the foreign gene and the above-mentioned tool of the known genome editing technique into the cells.

The cells may be cultured under appropriate culture conditions according to the cell type. If the foreign gene is a vector containing a drug resistance marker, the culture may be performed in the presence of the drug. By culturing in the presence of the drug, genome-edited cells can be efficiently selected. In addition, cells may be cloned by diluting or plating the cell culture medium.

[Analysis process]
In the insertion method of the present embodiment, after the insertion step, specifically, after introducing the foreign gene and the above-mentioned tool of the known genome editing technique into the cell, the foreign gene is placed in the upstream region of the Zxdb gene on the X chromosome of the cell. It may further include an analysis step of analyzing that the is inserted.

The analysis method is not particularly limited, but after the above insertion step, for example, after appropriately performing a culture step, the cell culture medium is plated, DNA is extracted from the resulting colonies, and the upstream region of the Zxdb gene on the X chromosome. A method of performing sequence analysis of the above can be mentioned.

≪Animal cells≫
In the animal cell of the present embodiment, the recombinase recognition sequence is inserted in the upstream region of the Zxdb gene on the X chromosome.

The insertion position of the recombinase recognition sequence is the same as the position exemplified as the insertion position of the foreign gene in the insertion method of the above embodiment.

Examples of the animal species and cell types from which the animal cells are derived include those similar to those exemplified in the insertion method of the above embodiment.

The animal cell of the present embodiment can be produced by using the insertion method of the above embodiment by replacing the foreign gene with the recombinase recognition sequence in the insertion method of the above embodiment.

Further, in the animal cell of the present embodiment, a foreign gene sandwiched between the recombinase recognition sequence may be inserted in the upstream region of the Zxdb gene of the X chromosome. Examples of the foreign gene include those similar to those exemplified in the insertion method of the above embodiment.

<Recombinase recognition sequence>
As used herein, the term "recombinase recognition sequence" refers to a recombinase recognition sequence in a so-called recombinase / recombinase recognition sequence system that utilizes a phenomenon in which a specific recombinase recognizes a recombinase recognition sequence and causes DNA recombination at that portion. means. Further, in the present specification, the term "base sequence" may mean a nucleic acid consisting of the base sequence.

Examples of the recombinase / recombinase recognition sequence system include Cre recombinase derived from Bacterophage P1, Cre / loxP system using a 34-base pair loxP sequence recognized by Cre recombinase, FLP recombinase derived from yeast, and FLP recombinase. Examples include the FLP / FRT system using the FRT sequence, the R recombinase derived from Zygosaccharomyces rouxii, and the R / RS system using the RS sequence recognized by the R recombinase. Therefore, examples of the recombinase recognition sequence used in the production method of the present embodiment include FRT sequence, loxP sequence, RS sequence and the like. Among them, the FRT sequence or the loxP sequence is preferable as the recombinase recognition sequence.

The FRT sequence includes, for example, 5'-GAAGTTTCATTTCTACGAAAAG.
A base sequence such as TATAGGAACTTC-3'(SEQ ID NO: 5) can be used.

As the loxP sequence, in addition to the base sequence represented by 5'-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3'(SEQ ID NO: 6), for example, Siegel RW, et al., Using an in vivo phagemid system to identify non-compatible loxP sequences, FEBS. 5'-ATAACTTCGTATAGTATACATTACGAAGTTATT-3'(lox511, SEQ ID NO: 7), 5'-ATAACTTCGTATAGGATACTTTTACGAAGTTATT-3'(lox2272, SEQ ID NO: 8), 5'ACTTAGT Variant lox such as -3'(loxFAS, SEQ ID NO: 9)
A P array or the like can be used.

As the RS sequence, for example, a base sequence such as 5'-TTGAATAGAAAGAATAACGTATTTTCATCATCAA-3'(SEQ ID NO: 10) can be used.

Even if a recombinase recognition sequence is present on the genome, recombination does not occur unless a specific recombinase is present. Therefore, by expressing the recombinase in a time-specific or tissue-specific manner, recombination by the recombinase can be caused in a time-specific or tissue-specific manner.

≪Kit for inserting foreign genes≫
The foreign gene insertion kit of the present embodiment (hereinafter, may be simply referred to as “the kit of the present embodiment”) includes the following.
Animal cells of the above embodiment;
A recombinase that recognizes the recombinase recognition sequence inserted into the chromosome of the animal cell;
A vector having the same recombinase recognition sequence as the recombinase recognition sequence inserted into the chromosome of the animal cell.

In the kit of the present embodiment, the combination of the recombinase and the recombinase recognition sequence may be the same as the combination exemplified in the animal cell of the above embodiment.

The recombinase may be in the form incorporated in the expression vector. Examples of the expression vector include the same as those exemplified in the insertion method of the above embodiment.

In the vector having the same recombinase recognition sequence as the recombinase recognition sequence inserted into the chromosome of the animal cell, a foreign gene sandwiched between the recombinase recognition sequences may be inserted. Examples of the foreign gene include those similar to those exemplified in the insertion method of the above embodiment.

According to the kit of the present embodiment, as shown in Examples described later, a desired foreign gene is easily inserted into the upstream region of the Zxdb gene of an animal cell by a recombinase-mediated cassette exchange method. The system to obtain can be provided.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Materials and methods]
(Culture of MIN6 cells)
MIN6 cells, a mouse-derived insulin-secreting cell line, were used in Dalveco-modified Eagle's Medium (DMEM) (Sigma) containing 15 w / v% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL strepmycin. It was cultured under the condition of 5% CO ₂ .

(Construction of plasmid)
Restriction enzymes, Quick Blending kit, One Taq DNA polymerase system from New England Biolabs Japan, Rapid DNA Dephos & Ligation Kit from Roche Digital

Two oligonucleotides (FRTmFRTw-F and FRTmFRTw-R) with a wild-type FRT sequence (FRTw) and a mutant FRT sequence (FRTm) are used to construct an acceptor that is integrated into the chromosome and receives an arbitrary expression unit. The synthesis was outsourced to Sigma-Aldrich Japan, and PCR was performed.

FRTmFRTw-F:
5'-GAGCTCGAAGTTCCTATTCCGAAGTTCCTATTCTTCAAATAGTATAGGAACTTCAAGCTTCCGAATTCAACTGCA-3' (SEQ ID NO: 11)
FRTmFRTw-R:
5'-GGTACCGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCTGCAGTTGAATTCGGAAGCT-3' (SEQ ID NO: 12)

These two oligonucleotides have complementary 3'sides, and by annealing each other and amplifying by PCR, SacI-FRTm-HindIII-EcoRI-PstI-reversed FRTw-KpnI (SacI, HindIII, etc.) can be used. , Representing the SacI restriction enzyme recognition sequence and the HindIII restriction enzyme recognition sequence, respectively). PCR was performed at 51 ° C and subcloned into pBluescript. Furthermore, rabbit β-globulin splice acceptor site (SA), the poly A signal region of herpes simplex virus thymidine kinase was used from pCAGGS, and the zeosin resistance gene was used from pFlip-in (manufactured by Life Technology). The DNA prepared by these (hereinafter, sometimes referred to as "expression unit") was integrated between the left and right homology arms of the Rosa26 locus of the pZDmR26 plasmid to construct a plasmid. When the expression unit is integrated into the chromosome and receives mRNA transcribed by either promoter in SA, it acquires zeocin resistance.

Donor plasmids for recombinase-mediated cassette exchange were similarly prepared. The cDNA of blastidin instead of zeocin was taken out from pcDNA6 (manufactured by Life Technology) and used. Furthermore, a unit expressing GFP was connected downstream of the TRE3G promoter. The TRE3G promoter was taken from the pTRE3G vector (manufactured by Clontech), and the rabbit β-globulin poly A region was taken out from pCAGGS to construct a GFP expression plasmid. GFP can be excised with SalI-EcoRI and replaced with any cDNA.
Yet another donor plasmid was prepared in the same manner and named the DsRed2 expression plasmid. When DNA is correctly inserted by this donor plasmid, it becomes resistant to hygromycin, and DsRed2 (manufactured by Clontech) causes the cells to glow red with the addition of doxycycline.
Yet another donor plasmid was prepared in the same manner and named the glucokinase expression plasmid. When DNA is correctly inserted by this donor plasmid, it becomes blastsidine-resistant, and β-cell glucokinase (cloned from MIN6c4 cell mRNA) mRNA is expressed by adding doxycycline.

(Gene transfer by electroporation method)
MIN6 cells were peeled off with trypsin EDTA, the number of cells was calculated, and then 2 × 10 ⁶ was collected by centrifugation. 100 μL of solution V supplied to Nucreator (manufactured by Lonza Japan) was taken, cells were suspended, 10 μg of a mixture of plasmid and mRNA was added, and the mixture was injected into a cuvette. DNA was introduced in the Nucreator setting G-016. The cells were suspended in DMEM and sorted with antibiotics from the 5th day.

(Insulin secretion test)
2 × 10 ⁵ MIN6 cells were seeded in each well of a 24-well plate, doxycycline was added to a final concentration of 1 μg / mL after 48 hours, and an insulin secretion experiment was performed after an additional 48 hours. Add 0.1 w / v% BSA (manufactured by Sigma) to Krebs-Ringer Bicarbonate buffer (KRB: Na ⁺ 144 mM, HCO 3-25 mM ^, Ca ₂ ⁺ 1.5 mM, pH 7.4) to make a secretory experimental solution, 1 M. Appropriate amounts of glucose solution were added to prepare 5 mM and 12.5 mM glucose solutions. The culture solution was aspirated from each well, washed once with KRB buffer, cultured in 5 mM glucose solution for 30 minutes at 37 ° C., replaced with 12.5 mM glucose solution, and cultured at 37 ° C. for 60 minutes. Insulin concentration was measured with a mouse insulin ELISA kit (manufactured by Shibayagi).

[Example 1]
(Analysis of insertion position on chromosome)
For the purpose of developing a method for efficiently producing a gene-introduced insulin-secreting cell line, MIN6 cells, which are insulin-secreting cell lines derived from mice, so that the gene to be introduced is inserted at a specific position on the chromosome. An acceptor sequence was placed on the chromosome. The acceptor sequence contains the FRT sequence and is capable of DNA exchange with a donor plasmid having a similar FRT sequence in the presence of Flip-recombinase (Flip).
First, the Rosa26 locus of the chromosome was selected at the insertion position, the acceptor sequence was inserted, and a plasmid containing green fluorescent protein (GFP) and a plasmid expressing Flip were introduced to induce an exchange reaction. Although the exchange reaction was considered successful, it was presumed that the expression of GFP was mosaic-like, which differed from cell to cell, and the average expression level was not high. Therefore, we investigated a method of selecting a clone that was inserted at an arbitrary position on the chromosome, inserted at the position of a chromosome with high transcriptional activity, and had high GFP expression after the exchange reaction. The specific method is shown below.

In the plasmid vector shown in FIG. 1, an expression unit sandwiched between acceptor sequences and having no promoter was cut out using a restriction enzyme, and a DNA fragment of 5 μg of the expression unit was introduced into MIN6 cells by an electroporation method. Randomly inserted into the genome.

On the 4th day after cell introduction, sorting was started at zeocin 200 μg / mL, or 400 μg / mL. It is considered that some clones that have become zeocin-resistant have one copy of the expression unit inserted, and some clones have several copies or dozens of copies introduced. In this case, even when a plurality of copies are inserted, in many cases, they are inserted vertically at the same position on the chromosome, and are rarely inserted at different positions on the chromosome. Therefore, when these zeosin-resistant clones, that is, the cells into which the expression unit is inserted, undergo recombinase-mediated cassette exchange with the expression unit containing the blastsidedin resistance gene, one copy is obtained in the cells in which the exchange has occurred successfully. Alternatively, MIN6 cells by electroporation of the GFP expression plasmid (see Figure 2) and the pCAG-Flppe plasmid into a pool of zeosin-resistant clones, assuming that they were exchanged with only a few copies in one place. Introduced to. Blasticidin was sorted at 2.5 μg / mL for 3 weeks after cell introduction.

As a result, 34 clones that became blastsaidin resistant were obtained. Of these, 29 clones were zeocin-sensitive, and expression of GFP was observed by induction with the addition of doxycycline. In addition, 5 clones were also resistant to zeocin, of which 3 clones showed GFP expression and 2 clones did not.

Among the 29 clones, the insertion positions of the expression units were identified using the genome walking kit (Clonech / Takara 636405, Universal Genome Walker ^TM 2.0) for the top 4 clones having uniform expression levels and high expression intensities.

As a result, the expression unit was inserted in the middle of the gene for 3 clones. On the other hand, it was revealed that one clone is located at the 94th, 713, 947th position on the X chromosome, that is, 10,622bp (about 10.6kbp) upstream from the transcription start site of the Zxdb gene on the X chromosome. (See Fig. 3).

[Example 2]
(Comparison of GFP expression due to difference in insertion position)
The expression unit containing the GFP gene obtained in Example 1 was inserted into the MIN6 cell at a position approximately 10 kbp upstream from the transcription start site of the Zxdb gene on the X chromosome, and the expression unit containing the GFP gene was inserted into the Rosa26 locus. MIN6 cells were observed under a microscope (KEYENCE All-in-One Fluorescense midroscope BZ-X710, magnification: 100 times) and compared. The results are shown in FIG.

As shown in FIG. 4, MIN6 cells in which an expression unit containing the GFP gene was inserted in the upstream region of the Zxdb gene on the X chromosome had a more uniform expression level of GFP than MIN6 cells inserted in the Rosa26 locus. Moreover, it was confirmed that the expression intensity was high.

[Example 3]
(Exchange of expression unit by recombinase-mediated cassette exchange method)
The DsRed2 expression plasmid (see FIG. 5) and the pCAG-Flpe plasmid were introduced into MIN6 cells in which the expression unit containing the GFP gene obtained in Example 1 was inserted into the upstream region of the Zxdb gene on the X chromosome by the electroporation method. Then, the recombinase-mediated cassette exchange was caused. Hygromycin was sorted at 200 μg / mL for 3 weeks after cell introduction.

As a result, clones that became resistant to hygromycin were obtained. Expression of DsRed2 was observed in these clones by induction with the addition of doxycycline (not shown).

[Example 4]
(Construction of insulin hypersecreting cells)
A glucokinase expression plasmid (see FIG. 6) and a pCAG-Flppe plasmid (manufactured by Addgene, #) were introduced into MIN6 cells in which the expression unit containing the DsRed2 gene obtained in Example 2 was inserted into the upstream region of the Zxdb gene on the X chromosome. 13787) was introduced by the electroporation method to cause recombinase-mediated cassette exchange. Blasticidin was sorted at 2.5 μg / mL for 3 weeks after cell introduction.

As a result, clones that became blastsaidin resistant were obtained. For these clones, the expression of glucokinase was induced by induction with the addition of doxycycline, and an insulin secretion test was performed. The results are shown in FIG.

As shown in FIG. 7, in the MIN6 cells in which the expression unit containing the glucokinase gene was inserted in the upstream region of the Zxdb gene of the X chromosome, the MIN6 cells into which the glucokinase gene was not introduced and the expression unit containing the glucokinase gene were found. The amount of insulin secreted was significantly higher than that of MIN6 cells inserted at the Rosa26 gene locus.

[Example 5]
(Insert into chromosome using CRISPR / Cas system)
4.0 μg of Cas9 expression plasmid, 3.5 μg of donor plasmid (see FIG. 8) and 2.5 μg of guide RNA expression plasmid (see FIG. 8) were introduced into MIN6 cells by the electroporation method using Nucleofector (manufactured by Lonza). Then, using the CRISPR / Cas system, a DNA fragment derived from the donor plasmid (a region containing the cDNA of zeosin) was inserted into the upstream region of the Zxdb gene on the X chromosome. The full-length base sequence of the donor plasmid is shown in SEQ ID NO: 13, and the base sequences of Left arm and Right arm in the donor plasmid are shown in SEQ ID NOs: 1 and 2, respectively. Further, the full-length base sequence of the guide RNA expression plasmid is shown in SEQ ID NO: 14, and the target sequence of the upstream region of the Zxdb gene of the X chromosome in the guide RNA expression plasmid is shown in SEQ ID NO: 4. In the guide RNA expression plasmid, the transcription efficiency of the U6 promoter was taken into consideration, and the sequence was prepared by adding one G (guanine) before the target sequence.

Then, in the presence of zeocin, the cells were cultured for 21 days, and the formed colonies were picked up in a cloning cylinder and seeded in a 24-well plate. When the number of cells increased, half was used for subculture and the other half was used for genomic DNA extraction.

PCR was performed on the extracted genomic DNA, and it was confirmed that a DNA fragment derived from the donor plasmid (a region containing the cDNA of zeocin) was inserted. The sequences of the primers used are shown below. In addition, the target position on the chromosome of the designed primer is shown in FIG.
op1222: 5'-GTGTCTAAGGGACAGACTTCCG-3'(SEQ ID NO: 15)
op1272: 5'-GGGAAACTTGGAAGTCAGTTGTCTGC-3'(SEQ ID NO: 16)
op1251: 5'-GAACGGCACTGGGTCAACTTG-3'(SEQ ID NO: 17)
op1252: 5'-GCAACTGCGTGCACTTCGTG-3'(SEQ ID NO: 18)

Then, for the colonies in which the DNA fragment derived from the donor plasmid was confirmed, a GFP expression plasmid (see FIG. 10) and a pCAG-Flppe plasmid (Adgene, # 13787) were used with Nucleofector (Lonza). It was introduced by the electroporation method and caused recombinase-mediated cassette exchange. Blasticidin was sorted at 2.5 μg / mL for 3 weeks after cell introduction.

As a result, clones that became blastsaidin resistant were obtained. For these clones, the expression of GFP was induced by induction with the addition of doxycycline. FIG. 11 shows a microscopic image of MIN6 cells in which an expression unit containing the GFP gene was inserted into the upstream region of the Zxdb gene on the X chromosome (microscope: KEYENCE All-in-One Fluorescence midroscope BZ-X710, magnification: 100 times, left. : Bright field image, right: Fluorescent image).

As shown in FIG. 11, it was confirmed that GFP was expressed almost uniformly.

According to the insertion method of the present embodiment, uniform expression of a foreign gene can be achieved.

Claims (12)

A method for inserting a foreign gene onto the chromosome of an animal cell, which comprises the step of inserting the foreign gene into the upstream region of the Zxdb gene of the X chromosome of the animal cell. The insertion method according to claim 1, wherein the upstream region of the Zxdb gene is a region 1 to 50 kbp upstream from the transcription start site of the Zxdb gene. The insertion method according to claim 1 or 2, wherein the upstream region of the Zxdb gene is a region 5 to 15 kbp upstream from the transcription start site of the Zxdb gene. The insertion method according to any one of claims 1 to 3, wherein the upstream region of the Zxdb gene is a region 9 to 11 kbp upstream from the transcription start site of the Zxdb gene. An animal cell in which a recombinase recognition sequence is inserted in the upstream region of the Zxdb gene on the X chromosome. The animal cell according to claim 5, wherein the recombinase recognition sequence is an FRT sequence or a loxP sequence. The animal cell according to claim 5 or 6, which is a cell derived from a human or a mouse. The animal cell according to any one of claims 5 to 7.
A recombinase that recognizes the recombinase recognition sequence inserted into the chromosome of the animal cell,
A vector having the same recombinase recognition sequence as the recombinase recognition sequence inserted into the chromosome of the animal cell,
A kit for inserting foreign genes. Foreign genes and
A nucleic acid having a sequence homologous to the upstream region of the Zxdb gene on the X chromosome of the target animal cell bound upstream and downstream of the foreign gene.
Including the vector. A nucleic acid having a sequence homologous to the sequence represented by SEQ ID NO: 1 is bound upstream of the foreign gene, and a sequence homologous to the sequence represented by SEQ ID NO: 2 is located downstream of the foreign gene. The vector according to claim 9, wherein the nucleic acid having the nucleic acid is bound. A guide RNA containing a nucleic acid consisting of the base sequence represented by SEQ ID NO: 3. A guide RNA expression vector containing a nucleic acid consisting of the base sequence represented by SEQ ID NO: 4.

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